Alzheimer's disease (AD) is characterized by the deposition of beta- amyloid (Abeta) into senile plaque, the formation of neurofibrillary tangles, and neuronal death. The Abeta protein, a 39 to 42/43 amino acid peptide is derived from the processing of the amyloid precursor protein (APP) by beta- and gamma- secretases. There are three known pathways of Abeta generation: a lysosomal/endosomal pathway, a trans-Golgi network pathway that generates mainly extracellular Abeta/1-40, and a endoplasmic reticulum/intermediate compartment pathway that results in the accumulation of intracellular Abeta/1-42. In contrast, APP processed by an alpha-secretase produces only a large secreted non-amyloidogenic derivative (APP-S/alpha). Physiological stimuli, such as muscarinic agonists and glutamate, increase the secretion of APP-S/alpha and decrease extracellular Abeta production, suggesting that pharmacological manipulation of this pathway may be of potential therapeutic benefit to decreased the beta-amyloid load. The long-term goals of this project are to understand in detail the molecular mechanisms involved in APP-S/alpha secretion from NT2N neurons, and their impact on intracellular and extracellular Abeta production. The signaling mechanisms involved in glutamate-induced APP-S/alpha secretion are poorly understood. Our preliminary studies show the presence of metabotropic glutamate receptors in NT2N cells. Glutamate stimulation of NT2N neurons causes an increase in intracellular calcium, activation of protein kinase C, and an increase in APP-S/alpha secretion appears to require ionotropic and metabotropic receptors, although the precise contribution of each receptor-associated signal transduction pathway in unclear. Furthermore, increasing intracellular calcium and/or activating protein kinase C with phorbol esters stimulates APP-S/alpha secretion. Based on our preliminary data, the hypothesis to be tested is that activation of protein kinase C mediates metabotropic glutamate-induced APP-S/alpha secretion, while calcium sensing by synaptotagmin mediates APP-S/alpha secretion induced by increased in intracellular calcium. The general strategy used, to dissect the components of the signaling pathways regulating APP-S/alpha secretion, is to express each potential component of the signaling pathway (glutamate receptor isotype, active or inactive protein kinase C isoform, active or inactive kinase substrate, active or inactive synaptotagmin) in NT2N neurons and measure APP-S/alpha secretion and intracellular and extracellular Abeta production.